A bioterrorist arsenal is likely to include a broad spectrum of bacterial species including the Category A pathogens Yersinia pestis (plague), a Gram-negative species, and Bacillus anthracis (anthrax), a Gram-positive species. Broad-spectrum antibiotics are potentially the most valuable for biodefense because these agents can be used quickly even in the absence of confirmatory diagnostic identification of species and can be stockpiled more efficiently and at lower cost. The overall goal of this application is to identify new antibacterial agents useful for biodefense against a broad spectrum of species and a wide range of drug resistance mechanisms. In Phase I, we will focus on a novel chemical series that potently inhibits UDP-GlcNAc enolpyruvyl transferase (EPT), an essential, broadly conserved, bacterial-specific enzyme catalyzing the first committed step in peptidoglycan synthesis. We will optimize this inhibitor series through a focused application of rational and structure-based drug design to generate lead compounds that are effective in an in vivo infection model. The specific aims are to (1) optimize the potency of the new inhibitors on EPT from Y. pestis (Yp) and B. anthracis (Ba); (2) demonstrate target specificity and inhibition of growth or viability of natural and engineered surrogates of Yp and Ba; and (3) demonstrate in vivo efficacy of EPT inhibitors against Yp and Ba surrogates in animal models of infection as well as determine in vitro ADMET profiles for the lead compounds. In Phase II, we will optimize an in vivo validated lead to the status of a clinical candidate. [unreadable] [unreadable]